Abstract Pseudomonas aeruginosa is a major cause of intensive care unit pneumonias and the number two cause of Gram-negative bacteremia and nosocomial pneumonia. Because of its ability to evade current antibiotics or develop resistance, P. aeruginosa clinical strains are increasingly resistant to all current clinically relevant antibiotics. Yet, the current pipeline of antibiotics in general, but anti-pseudomonal agents in particular, is alarmingly empty. Much of this failure is due to the incredible challenge of finding lead compounds against P. aeruginosa for further development because of its intrinsic barriers and resistance to small molecules. Herein, we propose a new method to identify such lead compounds that circumvent these barriers by taking an approach that interfaces genomics and novel high-throughput chemical screening technologies. Using genomics, we will identify essential outer membrane proteins (OMPs) that are valid targets for antibiotic discovery, thus circumventing the need for small molecule intracellular accumulation. We will then perform chemical screening in a multiplexed fashion against strains hypersensitized to inhibitors by controlled low expression of the respective OMP. This multiplexed approach will increase the efficiency and ability to identify small molecule leads for further development. In the R21 phase of the proposal, we will identify essential outer membrane proteins (OMPs) across many different strains of P. aeruginosa under clinically relevant growth conditions using recently developed genome-wide negative selection technology. Combining this dataset with publically available proteomic studies on OMPs, we will select a core set of essential OMPs to be targeted for small molecule discovery. Further, in the R21 phase, we will develop a method for Multiplexed Targeting of Essential Proteins (MTEP), which would allow simultaneous chemical screening against numerous essential targets. We will screen a small molecule library against a pool of bar-coded, genetically engineered target-specific screening strains in which each of the essential OMP genes has been knocked-down. This controlled low expression will confer hypersensitivity to small molecule inhibitors of the respective target. We will screen against 20 OMP targets simultaneously, in contrast to parallel individual screens against each of these strains, which rapidly becomes cost-prohibitive. This multiplexed strategy couples whole cell screening with target identification. Finally, in the R33 phase of the proposal, we propose to scale up MTEP screening against a large, unique collection of diversity-oriented synthetic (DOS) compounds, to identify candidates for further development as novel anti-pseudomonal antibiotics. Thus, we will develop a method for more efficiently identifying lead small molecules against the challenging highly resistant Gram-negative pathogen P. aeruginosa and will develop several candidate scaffolds for ultimate challenge in an animal model.